A crucial element in the development of effective vaccine strategies for AIDS is an experimental animal model in which the course of immunodeficiency virus infection parallels the pathogenesis of the human disease. SIV infection of macaques is a relevant model since it induces an immunodeficiency syndrome in infected macaques that is remarkably similar to human AIDS. VACCINE STUDIES: Our prior vaccine studies focused on the use of the highly attenuated modified vaccinia virus Ankara to express SIV proteins. Immunization with MVA-SIV recombinants resulted in a reduction in setpoint plasma viral load that was associated with prolonged survival. Better preservation of memory CD4+ T cells was correlated with titers of neutralizing antibodies to the challenge virus prior to challenge suggesting an important role for NAb and Envelope immunogens in this model. We therefore used a prime-boost, envelope-based approach as a strategy to generate neutralizing antibody responses. For this strategy we used a MVA recombinant expressing the neutralization-resistant SIVsmE543-3 envelope as the prime and trimeric SIVsmE543-3 envelope generated from a plasmid of codon-optimized gp140 as a boost. Rhesus macaques were immunized with either: A) Trimer alone, B) MVA-nonrecombinant, or C) MVA-Env prime, + trimer boost; all were given with adjuvant (AbISCO-100). All SIV Env immunized animals developed neutralizing antibodies to Tier 1 SIVsmE660 but only weak and partial neutralization of Tier 3 SIVsmE543-3 immunogen. They were challenged intrarectally with a single dose of SIVsmE660, 4 months following the last immunization. Four of six macaques that received prime-boost showed a 1-2 weeks delay of peak viremia accompanied with one log reduction in both peak and set point viremia when compared to control macaques vaccinated with non-recombinant MVA. The plasma viral load in these 4 macaques has been below detection limits from 18 weeks post challenge through 2-3 year of follow-up. Macaques vaccinated only with purified Env trimer uniformly showed earlier peak of viremia that was even higher than viral load shown by animal in control group. Despite this apparent enhanced acute viremia, two macaques from this group also were able to control viremia after an acute phase of infection. Control of viremia in these monkeys was associated with higher degree of preservation both central and effector memory subsets of CD4+ T cells and increased activation of B cells, as compared to non-controllers. Studies are underway to evaluate whether the breadth of neutralizing antibody responses in these animals correlates with control of viremia. NEUTRALIZING ANTIBODY RESPONSES AFTER SIV-INFECTION: Another focus of the lab has been the characterization of the breadth of neutralizing antibody responses after challenge with SIVsm. For these studies, we generated full length infectious clones of the challenge stock, SIVsmE660 by RT-PCR from the virus stock. Three of these viruses replicate efficiently in rhesus PBMC in vitro and were very sensitive to neutralizing antibody (equivalent to Tier 1 classification used for HIV-1. Two clones were evaluated for in vivo viral replication and pathogenesis rhesus macaques following intravenous inoculation. Both clones resulted in robust and persistent viremia. Evolution of the V1 and V4 regions was associated with escape from neutralization. We developed a panel of chimeric E660 clones expressing a range of late stage envelopes with varying sensitivity to neutralization cloned from plasma of these animals. Screening of these chimeric clones using post challenge sera from SIVsmE660-infected macaques allowed us to define at least three groups roughly equivalent to the Tiers 1, 2 and 3 described for HIV-1. We created and characterized representative Tier 1, Tier2 and Tier 3 variants from this panel and are currently evaluating their infectivity, viral replication and associated pathogenesis in rhesus macaques. The goal of this study is to determine whether the neutralization sensitivity of the infecting SIV influences viremia, generation of autologous and heterologous neutralizing antibody and/or disease progression. We infected rhesus macaques with a SIVsmE660 molecular clone and two representative autologous NAb escape variants. The molecular clone and its Nab escape variants have similar sequences but different sensitivity to NAbs (Tier 1A, Tier 2 and Tier 3). The three viruses had similar replicative fitness in rhesus macaques, as indicated by similar virus acquisition rate and plasma viremia during the acute phase of infection. A stepwise development of NAb responses to the three virus strains was observed in all animals, with Tier 1 viruses neutralized early, Tier 2 viruses somewhat later and Tier 3 viruses much later after infection. NAb development was similar regardless of which strain the macaques received. As a result, the selective pressure exerted by the NAbs against autologous viruses in the three cohorts differed, resulting in different viral divergence rates in the three groups as determined by NGS sequencing, with the most rapid divergence rate observed in the cohort infected with the Tier 1A virus. Consistent with a role for Nab responses in disease progression, the cohort infected with Tier 1 viruses showed a slower disease course as compared with those infected with Tier 3 viruses. These results provide direct evidence of the protective role of autologous NAb response in AIDS disease progression, and also suggest that NAb selective pressure should be used as one of the criteria for evaluation of HIV vaccine efficacy. CONTROL OF HETEROLOGOUS SIV WITH DNA/PROTEIN CO-IMMUNIZATION. We previously developed a method of simultaneous vaccination with DNA and protein resulting in high and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against SIV infection in macaques. To further optimize the DNA+protein co-immunization regimen, a SIVmac251-based vaccine formulated with either of two Toll-like Receptor 4 (TLR-4)-based liposomal adjuvant formulations (TLR-4+TLR-7 or TLR-4+QS21) was tested in macaques. Similar robust levels of humoral responses targeting the vaccine virus-matched SIVmac251 and heterologous SIVsmE660 were elicited by both vaccines, including antibodies recognizing V2. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in the vaccinees compared to controls, which reached statistical significance in animals with the Tripartite motif-containing protein 5alpha (TRIM-5) resistant allele. Vaccinees were preferentially infected by SIVsmE660 transmitted founder (T/F) virus carrying neutralization resistant A/K mutations at residues 45/47 in Env, demonstrating a strong vaccine-induced sieve effect. Delay in virus acquisition directly correlated with SIVsmE660-specific systemic neutralizing antibodies and mucosal gp70-V1V2 binding antibodies. These data support a combined contribution of immune responses and genetic background to vaccine efficacy. Following acquisition, reduction of viremia inversely correlated with humoral responses targeting V2 (cyclic V2, gp70-scaffolded V1V2) and SIV-specific T-cell responses. Although both DNA+protein vaccine groups showed delayed virus acquisition, the TLR4+7 adjuvanted vaccine induced stronger protective responses. In conclusion, combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced high, durable and potent cellular and humoral responses contributing to lower risk of infection by the heterologous SIV.